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Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia.

ABSTRACT: Propionic acidemia (PA) is a life-threatening disease caused by the deficiency of a mitochondrial biotin-dependent enzyme known as propionyl coenzyme-A carboxylase (PCC). This enzyme is responsible for degrading the metabolic intermediate, propionyl coenzyme-A (PP-CoA), derived from multiple metabolic pathways. Currently, except for drastic surgical and dietary intervention that can only provide partial symptomatic relief, no other form of therapeutic option is available for this genetic disorder. Here, we examine a novel approach in protein delivery by specifically targeting and localizing our protein candidate of interest into the mitochondrial matrix of the cells. In order to test this concept of delivery, we have utilized cell penetrating peptides (CPPs) and mitochondria targeting sequences (MTS) to form specific fusion PCC protein, capable of translocating and localizing across cell membranes. In vitro delivery of our candidate fusion proteins, evaluated by confocal images and enzymatic activity assay, indicated effectiveness of this strategy. Therefore, it holds immense potential in creating a new paradigm in site-specific protein delivery and enzyme replacement therapeutic for PA.

SUBMITTER: Darvish-Damavandi M

PROVIDER: S-EPMC4968140 | biostudies-literature | 2016 Sep

REPOSITORIES: biostudies-literature

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Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia.

Darvish-Damavandi Mahnaz M Ho Han Kiat HK Kang Tse Siang TS

Molecular genetics and metabolism reports 20160727

Propionic acidemia (PA) is a life-threatening disease caused by the deficiency of a mitochondrial biotin-dependent enzyme known as propionyl coenzyme-A carboxylase (PCC). This enzyme is responsible for degrading the metabolic intermediate, propionyl coenzyme-A (PP-CoA), derived from multiple metabolic pathways. Currently, except for drastic surgical and dietary intervention that can only provide partial symptomatic relief, no other form of therapeutic option is available for this genetic disorde ...[more]

PMID: 27504265

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Project description:Propionic acidemia is a rare metabolic disorder caused by a deficiency of propionyl- CoA carboxylase, the enzyme converting propionyl-CoA to methylmalonyl-CoA that subsequently enters the citric acid cycle as succinyl-CoA. Patients with propionic acidemia cannot metabolize propionic acid, which combines with oxaloacetate to form methylcitric acid. This, with the defective supply of succinyl-CoA, may lead to a deficiency in citric acid cycle intermediates.The objective of this study was to determine whether supplements with glutamine (400mg/kg per day), citrate (7.5mEq/kg per day), or ornithine ?-ketoglutarate (400mg/kg per day) (anaplerotic agents that could fill up the citric acid cycle) would affect plasma levels of glutamine and ammonia, the urinary excretion of Krebs cycle intermediates, and the clinical outcome in 3 patients with propionic acidemia.Each supplement was administered daily for four weeks with a two week washout period between supplements. The supplement that produced the most favorable changes was supplemented for 30 weeks following the initial study period and then for a 2 year extension.The urinary excretion of the Krebs cycle intermediates, ?-ketoglutarate, succinate, and fumarate increased significantly compared to baseline during citrate supplementation, but not with the other two supplements. For this reason, citrate supplements were continued in the second part of the study. The urinary excretion of methylcitric acid and 3-hydroxypropionic acid did not change with any intervention. No significant changes in ammonia or glutamine levels were observed with any supplement. However, supplementation with any anaplerotic agents normalized the physiological buffering of ammonia by glutamate, with plasma glutamate and alanine levels significantly increasing, rather than decreasing with increasing ammonia levels. No significant side effects were observed with any therapy and safety labs (blood counts, chemistry and thyroid profile) remained unchanged. Motor and cognitive development was severely delayed before the trial and did not change significantly with therapy. Hospitalizations per year did not change during the trial period, but decreased significantly (p<0.05) in the 2years following the study (when citrate was continued) compared to the 2years before and during the study.These results indicate that citrate entered the Krebs cycle providing successful anaplerotic therapy by increasing levels of the downstream intermediates of the Krebs cycle: ?-ketoglutarate, succinate and fumarate. Citrate supplements were safe and might have contributed to reduce hospitalizations in patients with propionic acidemia.

Project description:BackgroundPropionic acidemia (PA) is a disorder of intermediary metabolism with defects in the alpha or beta subunits of propionyl CoA carboxylase (PCCA and PCCB respectively) enzyme. We previously described a liver culture system that uses liver-derived hemodynamic blood flow and transport parameters to restore and maintain primary human hepatocyte biology and metabolism utilizing physiologically relevant milieu concentrations.MethodsIn this study, primary hepatocytes isolated from the explanted liver of an 8-year-old PA patient were cultured in the liver system for 10 days and evaluated for retention of differentiated polarized morphology. The expression of PCCA and PCCB was assessed at a gene and protein level relative to healthy donor controls. Ammonia and urea levels were measured in the presence and absence of amino acid supplements to assess the metabolic consequences of branched-chain amino acid metabolism in this disease.ResultsPrimary hepatocytes from the PA patient maintained a differentiated polarized morphology (peripheral actin staining) over 10 days of culture in the system. We noted lower levels of PCCA and PCCB relative to normal healthy controls at the mRNA and protein level. Supplementation of branched-chain amino acids, isoleucine (5mM) and valine (5mM) in the medium, resulted in increased ammonia and decreased urea in the PA patient hepatocyte system, but no such response was seen in healthy hepatocytes or patient-derived fibroblasts.ConclusionsWe demonstrate for the first time the successful culture of PA patient-derived primary hepatocytes in a differentiated state, that stably retain the PCCA and PCCB enzyme defects at a gene and protein level. Phenotypic response of the system to an increased load of branched-chain amino acids, not possible with fibroblasts, underscores the utility of this system in the better understanding of the molecular pathophysiology of PA and examining the effectiveness of potential therapeutic agents in the most relevant tissue.

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Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia.

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Towards the development of an enzyme replacement therapy for the metabolic disorder propionic acidemia.

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